The overall goal of this proposed research is to explore a preliminary observation of non-clonal evolution in cancer metastasis. Breast cancer is most frequently diagnosed cancer in women (except skin cancer) and the second leading cause of cancer death after lung cancer. Breast cancer arises from the epithelial cells of the breast ductal tree. We have previously developed several somatic mouse models of breast cancer evolution by adapting the RCAS-TVA method for introducing oncogenes into somatic tissues. This model also allows us to genetically track the small number of breast epithelial cells that suffered the initial oncogenic mutation in a field of fully developed mammary gland in vivo. Using this method to introduce either Wnt1 or ErbB2 to initiate the formation of triple-negative and HER2 breast cancer subtypes, the two most aggregative cancer subtypes of women, we found surprising evidence of non-clonal evolution of cancer. We found that within the resulting mammary tumor, there exists a minor population of cells that histologically appears to be carcinoma cells, but surprisingly lacks the provirus DNA and the initiating oncogene. Furthermore and even more unexpectedly, we found that many of the metastatic foci in the lungs also lack the provirus DNA and its oncogenic protein product. These unexpected preliminary data suggest a provocative hypothesis that as the initially mutated breast epithelial cells evolve to cancer, the neighboring normal breast epithelial cells that did not suffer the initiating oncogenic mutation can be instigated by the mutated cells to become cancerous and even metastatic. To test this non-clonal hypothesis for breast cancer metastasis, we will pursue three aims: Specific Aim 1: To verify our preliminary mouse model finding that the normal mammary epithelial cells can be stimulated by adjacent oncogene-activated mammary cells to undergo tumorigenesis and metastasis. Specific Aim 2: To determine whether normal human breast epithelial cells can be instigated by admixed human premalignant or cancerous cells to undergo tumorigenesis and metastasis. Specific Aim 3: To define the unique expression changes in the tumor cells that lack the initiating oncogene.